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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

• the invention relates to tools for expression of heterologous genes in Yarrowia lipolytica.
• the yeast Y. lipolytica is increasingly used as a host for the expression of genes of interest; in this context, so-called "integrative" vectors are used, which allow the insertion of a segment of DNA carrying the gene of interest into chromosomal DNA.
• integration vectors are used, which allow the insertion of a segment of DNA carrying the gene of interest into chromosomal DNA.
• Application EP 138 508 describes the transformation of Yarrowia lipolytica using vectors capable of integrating into chromosomal DNA by recombination of sequences of Y. lipolytica carried by said vectors, with the homologous sequences present on l Chromosomal DNA of the host cell.
• zeta sequences which correspond to LRTs (long terminal repetitions) of the Yltl retrotransposon of Yarrowia lipolytica; these sequences have been described by SCHMID-BERGER et al. [J. Bacteriol., 2477-2482 (1994)] which indicate that they are present in a large number of copies (approximately 35 copies of the complete retrotransposon, and approximately 30 copies of the isolated zeta sequence) in the chromosomal DNA of certain strains of Yarrowia lipolytica.
• the subject of the present invention is a method of integrating a gene of interest into the genome of a Yarrowia strain, using a recombinant vector carrying an insert flanked by zeta sequences and comprising said gene. interest, which method is characterized in that said recombinant vector is used to transform a strain of Yarrowia whose genome is devoid of zeta sequences.
• Yarrowia lipolytica strains lacking zeta sequences which can be used for implementing the process according to the invention are, for example, strains derived from the strain W29 (ATCC 20460 or CLIB 89, MatA), such as W29ura3-302 (CLIB 141, MatA Ura3-302), POla (CLIB 140, MatA Ura3-302, Leu2-270) and POld (CLIB 139, MatA Ura3-302, Leu2-270, xpr2-322), described by BARTH and GAILLARDIN [The di orphic fungus Yarrowia lipolytica. In: Non conventional yeasts in biotechnology (Wolf K. Ed.).
• the insert comprising the gene of interest further comprises sequences allowing the control of the expression of said gene and / or at least one marker for selection of the transformants.
• the selection marker consists of a gene whose expression allows the selection of transformants. It may for example be a gene for resistance to an antibiotic, or a defective mutant of a gene necessary for the growth of yeast, such as URA3, LEU2, etc.
• a selection marker will be chosen which needs to be present in several copies to be functional, which makes it possible to select the transformants having integrated several copies of the gene of interest.
• a defective URA3 marker which derives from the URA3 gene of Y. lipolytica allowing the complementation of auxotrophy for uracil, such as the URA3d markers described by LE DALL et al. [Curr. Broom. , 26, 38-44 (1994)].
• the expression control sequences are in particular promoter and terminator sequences active in Yarrowia.
• An inducible or constitutive promoter can be used.
• the promoters AC02 and LJP2 are both inducible by triglycerides and fatty acids.
• Other selection markers and control sequences which can be used for implementing the present invention are, for example, those cited by BARTH and GAILLARDIN (abovementioned publication).
• said insert further comprises signals for controlling the secretion of said product.
• Functional signal sequences can be used for this purpose in Yarrowia lipolytica, for example all or part of the prepro sequence of the LIP2 gene described in the French Application in the name of LABORATOIRES MAYOLY-SPINDLER, mentioned above.
• the present invention also relates to transformed Yarrowia cells capable of being obtained by the process according to the invention.
• these transformed cells comprise at least 2 copies of the insert flanked by zeta sequences, comprising the gene of interest, integrated into their genome in a dispersed manner.
• the present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples of obtaining transformed strains of Yarrowia lipolytica in accordance with the invention.
• EXAMPLE 1 CONSTRUCTION OF VECTORS INCLUDING A ZETA SEQUENCE
• vectors are obtained from the vector pINA1067, derived from a vector of the pINA970 type described by BARTH and GAILLARDIN (abovementioned puolication, FIG. 6), by excision of the portion of sequence between the promoter and the XPR2 terminator, which has and has been replaced by a linker containing the restriction sites SrfI and B_gJ.II.
• pINA1067 carries the defective URA3 marker ura3d4 described by LE DALL et al. (publication cited above), as well as a tetracycline resistance marker.
• the plasmid pHSS6 carrying an origin of replication for E.
• coli and a kanamycme resistance gene is linearized by restriction with NotI and introduced into the NotI site of pINA1067.
• the ligation mixture is used to transform E. coll.
• the transformants having integrated the plasmid are selected on tetracycline (6 mg / 1) and kanamycme (40 mg / 1).
• JMP3 The resulting vector, designated JMP1, is cut with HindIII and EcoRI, which eliminates the fragment carrying the promoter and terminator sequences of XPR2 and the marker for resistance to tetracycline; this fragment is replaced by a multisite linker which contains the restriction sites HindIII, ClaI, MluI, Hpal, BamHI and EcoRI.
• the resulting vector is called JMP3.
• the different stages of the construction of JMP3 are shown diagrammatically in Figure 1.
• the autocloning vectors JMP3 and JMP5 integrate into the genome after cleavage by the restriction enzyme Notl. This restriction allows the elimination of the bacterial part (origin of replication of E. coli and of the KanR resistance marker).
• Expression vectors comprising a sequence coding for the acid-resistant extracellular lipase of Yarrowia lipolytica, described in the French Application in the name of LABORATOIRES MAYOLY- SPINDLER, mentioned above, under the control of the promoter AC02 (vector JMP6) or of its own promoter (vector JMP10) have been constructed.
• JMP6 vector JMP6 vector
• the promoter of the acyl CoA oxidase AC02 gene was amplified by PCR from the total genomic DNA of Y. lipolytica, with the oligonucleotides Aco2P1. and Aco2P2 which respectively contain a ClaI site and a HindIII site.
• Aco2Pl 5 'GCG ATCGAT CATACTGTTACACTGCTCCG
• the 2168 bp PCR fragment was cloned at the EcoRV site of the BLUESCRIPT KS + vector.
• the resulting plasmid is called KS-AC02prom.
• This vector was digested with ClaI and partially with HindIII to obtain a fragment of 2155 bp containing the complete AC02 promoter.
• a 2030 bp NdeI * -HindIII fragment containing the promoter of the Y. lipolytica lipase gene was isolated from the plasmid called pKS + -LIP2prom.
• the plasmid pKS + -LIP2prom was digested with NdeI, then treated with T4 DNA polymerase to make the site
• Ndel franc Ndel *
• NdeI * NdeI *
• HindIII The 2030 bp NdeI * -HindIII fragment was ligated with the 1134 bp HindIII-EcoRI fragment carrying the lipase gene and its terminator, and the ligation product inserted at the Hpal-EcoRI sites of the JMP3 vector.
• the resulting plasmid is called JMP10.
• Figure 2 shows the plasmids JMP6 and JMP10.
• EXAMPLE 3 OBTAINING TRANSFORMANTS OF Y. LIPOLYTICA.
• the plasmids JMP6 and JMP10 previously linearized with NotI, which makes it possible to eliminate the sequences originating from the plasmid pHSS6, are introduced into the cells of Yarrowia by transformation with lithium acetate, according to the protocol described by BARTH and GAILLARDIN.
• Usually 20 to 50 transformants / ⁇ g of DNA are obtained with the POld strain and 100 to 200
• transformants will be designated below according to the following nomenclature: name of the strain - number of the plasmid - number of the transformant.
• the structure of the transformants was studied by Southern blot.
• the total genomic DNA of the original strain (T) and of the different transformants is digested with HindIII in the case of POld and with BamHI (for the transformants JMP6) or EcoRI (for the transformants
• the probes used are derived from the zeta sequences, from the sequence of the promoter AC02, and from the sequence coding for the lipase LIP2.
• FIG. 3A represents the results observed with the lipase probe
• FIG. 3B represents the results observed with the ZETA probe
• FIG. 3C represents the results observed with the AC02 probe.
• FIG. 4A represents the results observed with the lipase probe + the AC02 probe
• FIG. 4B represents the results observed with the ZETA probe.
• FIG. 4A an amplification of the vector sequences is observed in the transformants, revealed by the intensity of the BamHI band of 2.9 kb (promoter AC02 + lipase gene) or of the EcoRI band of 1.6 kb (lipase gene), compared with that of the BamHI genomic band of 2.6 kb which corresponds to the AC02 genomic promoter, or that of the EcoRI genomic band of approximately 6 kb, which corresponds to the lipase gene.
• FIG. 4B we observe in strain E150, and the transformants, several zeta sites revealed by numerous bands in the EcoRI restriction profile. Integration in tandem is revealed by the intensity of the BamHI band of 2.5 kb, and the EcoRI band of 3.7 kb, corresponding to the fragments expected if the vector sequences are integrated in tandem.

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• 1998
  • 1998-09-01 FR FR9810900A patent/FR2782733B1/fr not_active Expired - Fee Related
• 1999
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  • 1999-09-01 PT PT99940267T patent/PT1108043E/pt unknown
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